Countercurrent distribution (CCD) was invented as a purification method in the 1940's based on the partitioning of solutes between two immiscible solvents [1]. The method does not require any solid-matrix support, and therefore offers the important advantages of minimizing losses of solutes due to adsorption to solid chromatographic matrices and preventing contamination by impurities from such matrices during the process of purification. However, because it requires multiple equilibrations and transfers of upper and lower solvent phases between a large battery of tubes, the method is mechanically cumbersome to implement. Accordingly, a number of modifications of CCD have been proposed to improve the methodology.
In the countercurrent chromatographic system called droplet countercurrent chromatography (DCCC) [2, 3], a mixture of solutes can be separated in a series of columns where a mobile phase is passed through a stationary phase droplet by droplet to bring about phase equilibration and solute exchange between the two phases. Since DCCC has no need for a solid chromatographic matrix, it does not incur any loss of solutes through irreversible adsorption to solid matrices, thus allowing recovery of practically all the solutes after evaporation of the solvents. DCCC also can work under room temperature and pressure, avoiding the need for expensive high pressure systems. Whereas systems such as gas chromatography and high performance liquid chromatography would entail loss of resolution due to non-optimal flow dynamics and surface adsorptions, DCCC is free of such problems because both phases in the system are liquids. However, as described in U.S. Pat. No. 3,853,765 [3], the diameter of the columns employed in DCCC is of the order of only 1.8 mm to make possible the droplet-by-droplet flow of the mobile phase, which greatly restricts the operable volume. Therefore separation by DCCC is mainly limited to analytical applications. For this reason, in recent years, usage of DCCC has been largely displaced by centrifugal countercurrent techniques.
Centrifugal countercurrent techniques include centrifugal partition chromatography (CPC) [4, 5], high speed countercurrent chromatography (HSCCC) [6-9] and high performance countercurrent chromatography (HPCCC) [10], where separation of the two immiscible liquid phases is speeded up by centrifugal force. The main disadvantage of these techniques is that the need for centrifugation limits the scale up process, because the separation columns/tubing have to fit into a centrifuge. After each separation, the solvents have to be flooded out by pumping nitrogen into the column, and this takes time and requires pressurized nitrogen. The requirement for centrifugation also has to be met with expensive equipment.
In another modification, namely controlled-cycle pulsed liquid-liquid chromatography (CPLC), mixing of upper and lower phases is conducted in columns segmented into a cascade of chambers by horizontal perforated plates, and equilibration of solutes between the phases needs to be achieved on a discontinuous basis with pauses for phase separation in between [11]. Its discontinuous mode of operation incurs mechanical inconvenience as in the original CCD procedure, and potential loss of resolution.